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The ability of apolipoprotein E (apoE) to be spared degradation in lysosomes and to recycle to the cell surface has been demonstrated by our group and others, but its physiologic relevance is unknown. In this study, we characterized apoE recycling in primary murine macrophages and probed the effects of HDL and apoA-I on this process. In cells pulsed with 125 I·apoE bound to VLDL, intact apoE was found in the chase medium for up to 24 h after the pulse. Approximately 27 ؎ 5% of the apoE internalized during the pulse was recycled after 4 h of chase. Addition of apoA-I and HDL increased apoE recycling to 45 ؎ 3% and 46 ؎ 3%, respectively, similar to the amount of apoE recycled after pulsing the cells with 125 I·apoE·HDL. In addition, apoA-I-producing macrophages from transgenic mice showed increased apoE recycling at 4 h (38 ؎ 3%). Increased ABCA1 expression potentiated apoE recycling, suggesting that recycling occurs via ABCA1. Finally, in the presence of apoA-I, recycled apoE exited the cells on HDL-like particles. These results suggest that apoE recycling in macrophages may be part of a larger signaling loop activated by HDL and directed at maximizing cholesterol losses from the cell. Apolipoprotein E (apoE) is a 34 kDa glycoprotein found on all plasma lipoproteins except the smallest LDLs. The primary extracellular function of apoE is to serve as a ligand for receptor-mediated uptake of lipoproteins through the LDL receptor, the LDL receptor-related protein, and heparan sulfate proteoglycans (1, 2). ApoE also plays a key role in intracellular lipid metabolism, influencing processes such as the assembly and secretion of lipoproteins (3-6), intracellular routing of endocytosed remnant lipoproteins (7, 8), and cholesterol efflux to HDL (9, 10). In macrophages, the effect of apoE on cholesterol efflux, as well as its other pleiotropic effects (2, 11, 12), may be critical in protecting the artery wall from atherosclerotic lesion formation (13-16).We and others have established that a portion of the apoE internalized by cells via lipoprotein receptors escapes lysosomal degradation and is recycled. This recycling is not cell specific, as it occurs in hepatocytes (17-20), fibroblasts (21, 22), hepatoma cells (21-25), and macrophages (19,26,27). The physiologic relevance of apoE recycling is unknown. Studies in our laboratory have shown that apoE recycling in hepatocytes is stimulated by apoA-I. Heeren et al. (22) showed that HDL increased the recycling of apoE as well as the efflux of cholesterol in fibroblasts using double-label experiments with 125 I-labeled apoE and 3 H-labeled cholesterol. It is also known that apoA-I stimulates the secretion of endogenous apoE by macrophages (28). Based on these observations, we speculated that apoE recycling is linked to reverse cholesterol transport both by serving as a signaling mechanism for HDL cholesterol entry into the cell and by increasing intracellular cholesterol efflux in response to HDL signaling (19). If apoE recycling is associated with cholesterol efflux ...

Obese mice without leptin (ob/ob) or the leptin receptor (db/db) have increased plasma HDL levels and accumulate a unique lipoprotein referred to as LDL/HDL1. To determine the role of apolipoprotein A-I (apoA-I) in the formation and accumulation of LDL/HDL1, both ob/ob and db/db mice were crossed onto an apoA-I-deficient (apoA-I ؊ / ؊ ) background. Even though the obese apoA-I ؊ / ؊ mice had an expected dramatic decrease in HDL levels, the LDL/HDL1 particle persisted. The cholesterol in this lipoprotein range was associated with both ␣ -and ␤ -migrating particles, confirming the presence of small LDLs and large HDLs. Moreover, in the obese apoA-I ؊ / ؊ mice, LDL particles were smaller and HDLs were more negatively charged and enriched in apoE compared with controls. This LDL/ HDL1 particle was rapidly remodeled to the size of normal HDL after injection into C57BL/6 mice, but it was not catabolized in obese apoA-I ؊ / ؊ mice even though plasma hepatic lipase (HL) activity was increased significantly. The finding of decreased hepatic scavenger receptor class B type I (SR-BI) protein levels may explain the persistence of LDL/HDL1 in obese apoA-I ؊ / ؊ mice. Our studies suggest that the maturation and removal of large HDLs depends on the integrity of a functional axis of apoA-I, HL, and SR-BI. Moreover, the presence of large HDLs without apoA-I provides evidence for an apoA-I-independent pathway of cholesterol efflux, possibly sustained by apoE. Unlike apolipoprotein B (apoB)-containing lipoproteins, which are formed by hepatocytes and enterocytes, HDL biogenesis is quite complex and involves mostly extracellular events (1, 2). In the intestine, apoA-I is secreted in association with chylomicrons. Once in the circulation, apoA-I is transferred to HDL in exchange for apoE and the C apolipoproteins. In the liver, apoA-I is secreted as part of nascent cholesterol-poor HDL particles. Additionally, HDL can be formed with excess surface material from remnant lipoproteins during hydrolysis. Circulating HDL is acted upon by many different enzymes, including HL and LCAT, leading to a complex catabolic fate, as its cholesteryl esters can be transferred to triglyceride (TG)-rich lipoproteins via cholesteryl ester transfer protein or be selectively taken up by the hepatic HDL receptor, scavenger receptor class B type I (SR-BI) (3), without internalization of the HDL particle. However, holoparticle uptake of large apoE-enriched HDLs has also been reported (4).Unlike their human counterparts, mouse models of obesity, such as the leptin-deficient and leptin receptordeficient mice (ob/ob and db/db, respectively), have increased levels of HDL without an increase in VLDL levels (5, 6). This increase in HDL makes these animals particularly resistant to the development of atherosclerotic lesions (7). Recent work by Tall and colleagues (6,8) has shown that the increase in HDL is attributable to both typical HDLs and larger HDLs (LDL/HDL1), and is caused by decreased hepatic HDL uptake in these animals. This LDL/HDL1 particle is...

Previous studies have demonstrated that macrophage-derived apolipoprotein E (apoE) reduces atherosclerotic lesion formation in lean apoE-deficient ((-/-)) mice. apoE has also been demonstrated to play a role in adipocyte differentiation and lipid accumulation. Because the prevalence of obesity has grown to epidemic proportions, we sought to determine whether macrophage-derived apoE could impact atherosclerotic lesion formation or adipose tissue expansion and inflammation in obese apoE(-/-) mice. To this end, we transplanted obese leptin-deficient (ob/ob) apoE(-/-) mice with bone marrow from either ob/ob;apoE(-/-) or ob/ob;apoE(+/+) donors. There were no differences in body weight, total body adipose tissue, or visceral fat pad mass between recipient groups. The presence of macrophage-apoE had no impact on adipose tissue macrophage content or inflammatory cytokine expression. Recipients of apoE(+/+) marrow demonstrated 3.7-fold lower plasma cholesterol (P < 0.001) and 1.7-fold lower plasma triglyceride levels (P < 0.01) by 12 wk after transplantation even though apoE was present in plasma at concentrations <10% of wild-type levels. The reduced plasma lipids reflected a dramatic decrease in very low density lipoprotein and a mild increase in high-density lipoprotein levels. Atherosclerotic lesion area was >10-fold lower in recipients of ob/ob;apoE(+/+) marrow (P < 0.005). Similar results were seen in leptin receptor-deficient (db/db) apoE(-/-) mice. Finally, when bone marrow transplantation was performed in 4-mo-old ob/ob;apoE(-/-) and db/db;apoE(-/-) mice with preexisting lesions, recipients of apoE(+/+) marrow had a 2.8-fold lower lesion area than controls (P = 0.0002). These results demonstrate that macrophage-derived apoE does not impact adipose tissue expansion or inflammatory status; however, even very low levels of macrophage-derived apoE are capable of reducing plasma lipids and atherosclerotic lesion area in obese mice.

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